Spray immunization of fish

ABSTRACT

A method for immunizing fish against disease by spraying with vaccine or bacterin.

This is a continuation of application Ser. No. 769,128, filed Feb. 16,1977, now abandoned.

BACKGROUND OF THE INVENTION

A number of vaccines have been developed for the control of diseases infish, as illustrated for example, in U.S. Pat. Nos. 3,492,400 to Klontzand 3,862,313 to Fryer et al. In the past such vaccines have beenadministered by peritoneal injection or by incorporation in food orwater. More recently vacuum infiltration (rapid pressure change) andhyperosmotic vaccine dips have been proposed. Peritoneal injections oflarge antigenic masses have been highly effective; however, when appliedto large numbers of fish this method is cumbersome and costly.Incorporation of vaccine into the food or water is probably the mostdesirable system since it is relatively simple and economical. However,its effectiveness has been demonstrated in only a limited number ofdiseases. In addition the duration of protection by this method has beenmuch shorter than that obtained by injection. The newer techniques ofvacuum infiltration and hyperosmotic vaccine dips lend themselves tovaccination of small fish, but would be time consuming and costly whenapplied to larger fish. Unless the disease causing organism isconstantly present to reinforce immunity, or if for any reason immunityis diminished, re-vaccination of larger fish with these techniques wouldbe inefficient.

Spray vaccination will be especially useful where previously vaccinatedfish are not constantly exposed to the disease causing agent. In thissituation, unreinforced immunity may significantly decrease with time.For example, if salmon are vaccinated against V. anguillarum as fry,hatchery rearing periods as long as one year or more without naturalcontact with the target agent may lead to a reduction in protection.Before releasing the fish from the hatchery, the spray technique of thisinvention could be employed to revaccinate them, or possibly to supply abooster shot.

The present invention comprises an antigenic material useful forimmunizing fish against disease and a spray method for administering theantigenic material to the fish.

Accordingly it is an object of the invention to provide an antigenicmaterial for the immunization of fish against disease.

Another object of the invention is to provide a method for administeringthe antigenic material to fish efficiently and at low cost.

Another object is to provide a method for administering the antigenicmaterial to the fish by means of a spray.

A further object of the invention is to provide a pressure spray methodfor immunizing fish employing antigenic material containing anabsorbent.

Yet a further object of the invention is to employ an antigenic materialcontaining an abrasive for administration to fish by means of a pressurespray.

DESCRIPTION OF THE INVENTION

Basically, our invention consists in removing the fish from water by adip net, and while suspended in the air spraying it with antibodymaterial. Another method is to cause the fish to pass through a troughand to spray the fish as they pass through. Ordinary spray orsandblasting guns may be employed to deliver the spray. The air pressureused must be sufficient to cause the spray to penetrate and to producewithin the body of the fish the humoral antibody response characteristicof the vaccine or bacterin. Ordinarily any pressure up to 90 psi issatisfactory, with the higher portion of a range of about 80 to 90 psibeing more desirable. Too high a pressure is to be avoided since it maycause physical damage.

The antigenic material employed may consist of exotoxins, killed, orattenuated microorganisms. We have found bacterins comprising formalinkilled bacteria to be highly useful, but other vaccines and bacterinsmay be employed.

EXAMPLE 1

A labelled vaccine of formalin killed Vibrio anguillarum bacterinattached to yellow fluorescent dye, consisting of 100 milligrams dryweight killed V. anguillarum (LS-1-74) per 1 gram dye was employed inthis experiment. The dye and wet bacteria was thoroughly mixed,lyophilized overnight at 100 micro Hg pressure, and ground in a mortar.A fifty fish group of coho salmon (Oncorhynchus kisutch) at 30/lb. weresuspended in a dip net and sprayed with the dry powder bacterin at adistance at 12-15 inches to coat the fish. As a control, a similar groupof coho salmon was sprayed with a blue fluorescent dye which containedno bacterin. Both groups were held at 13° C. for 27 days. Individualtiters against V. anguillarum (LS-1-74) were determined employing themicro titer technique against 5 fish in each group. A geometric meantiter of 1:196 was found in the vaccinated fish and zero titer in theunvaccinated group. The remaining fish in each group were exposed to anartificial water borne challenge to V. anguillarum LS-1-74. An 86%mortality resulted in the unvaccinated group and only 2% mortality inthe vaccinated group.

EXAMPLE 2

A series of tests were run to examine various parameters of the sprayvaccination technique. Different concentrations of formalin killed V.anguillarum bacterin were attached to fluorescent dye, as described inExample 1, the concentrations ranging from 1 microgram to 100 milligramsbacteria/gram dye.

Also, employed were water dispersions of the bacterin/dye formulation, akilled TGY culture of V. anguillarum, and a bacterin mixture withalumina.

Groups of 50 cold brand marked coho salmon were sprayed at 90 psi witheach formulation, and one group was sprayed with a dye preparationwithout bacterin as a control.

Table 1 gives the geometric mean of the individual titers and themortality suffered by the sample.

                  TABLE 1                                                         ______________________________________                                        Comparison of effectiveness of various concentrations                         and compositions of Vibrio anguillarum LS-1-74 bacterin spray                 preparations.                                                                                     Geometric                                                                     mean                                                                          individual                                                                    titers     Mortality suffered                                                 against    in an artificial                                                   Vibrio     challenge to                                                       anguillarum                                                                              Vibrio -  LS-1-74 (2 × 10.sup.6                                         bacteria/                                           Preparation    (five fish ml in challenge                                Run  Constitutents  sample)    tank)                                          ______________________________________                                        1    Blue fluorescent                                                              dye Control    1:2        37/47 79%                                           50:50                                                                         H.sub.2 O + 1 microgram                                                       bacterin per gram                                                        2    of yellow fluore-                                                             scent dye      1:5        24/43 56%                                           10 microgram                                                                  bacterin per gram                                                        3    of yellow fluore-                                                             scent dye      1:21       31/42 74%                                           100 microgram                                                                 bacterin per gram                                                        4    of yellow fluore-                                                             scent dye      1:21       25/45 56%                                           1 milligram                                                                   bacterin per gram                                                        5    of yellow fluore-                                                             scent dye      1:32        3/50 6%                                            10 milligram                                                                  bacterin per gram                                                        6    of yellow fluore-                                                             scent dye      1:147       0/45 0%                                       ______________________________________                                             10 milligram                                                             7    bacterin per gram                                                             of alumina     1:147       1/46 2%                                            100 milligram                                                                 bacterin per gram                                                        8    of yellow fluore-                                                             scent dye      1:256       0/45 0%                                            50:50                                                                         H.sub.2 O + 100 milli-                                                        gram bacterin                                                            9    per gram of yellow                                                            fluorescent dye                                                                              1:256       0/47 0%                                            96 hr., 30° C. TGY                                                     culture of v.                                                                 anguillarum LS-1-                                                        10   74 killed with 0.3%                                                           formalin       1:48        2/44 5%                                       ______________________________________                                    

The bacterin of Run 10 was prepared by innoculating a Trypton GlucoseYeast (TGY) broth with V. anguillarum LS-1-74 and allowed to grow at 30°C. for 96 hours. 0.3% formalin was added and the culture allowed tostand overnight. The preparation was checked for sterility and thensprayed on a group of 50 coho salmon. Table 1 shows that the TGY sprayedfish had an antibody titer greater than the 1 mg/gm dye preparation ofRun 5 and had a similar protection percentage.

A comparison of Runs 6 and 7 shows that the substitution of alumina foryellow fluorescent dye did not affect the results.

EXAMPLE 3

This experiment was carried out to determine the titers elicited byspraying polyvalent whole cell bacteria. Each vaccine tested consistedof 0.3% formalin killed Aeromonas salmonicida AS-SS-70, V. anguillarumLS-1-74 and V. anguillarum MSC-2-75. The spray preparations consisted of500 ml. deionized water, 50 grams of ground pumice (grade FFF) and 1gram wet-weight of each included bacterin type. Approximately 400micrograms (dry weight) of each included bacterin per ml. was theconcentration. The purpose of the pumice was to scarify the fish withthe hope of thereby increasing the titer. Four vaccines were preparedconsisting of AS-SS-70, AS-SS-70 and LS-1-74, AS-SS-70 plus LS-1-74 andMS-275, and LS-1-74 plus MSC-2-75. Fish samples of 50 coho salmon eachwere sprayed with each of the four vaccines and with a controlcontaining no bacterin. By calculation, each individual fish receivedabout 0.5 ml of preparation. The fish were held at 13° C. for 29 days.Titers were then taken on five individual fish from each group. Theresults are shown in the following table.

                                      TABLE 2                                     __________________________________________________________________________    Titers against constituent antigens of various combinations of                bacterins applied by spray injection. All titers reported are                 geometric means of individual determinations on five fish samples.                                      AS-SS-70                                                   NONE         AS-SS-70                                                                            LS-1-74                                                                             LS-1-74                                       Titer Antigen                                                                        CONTROL                                                                              AS-SS-70.sup.1                                                                      LS-1-74.sup.2                                                                       MSC-2-75.sup.3                                                                      MSC-1-75                                      __________________________________________________________________________    AS-Sil-67.sup.4                                                                      1:55   1:64  1:64  1:147 1:64                                          LS-1-74                                                                              0      0     1:16  1:28  1:16                                          MSC-2-75                                                                             0      0     0     1:169 1:64                                          __________________________________________________________________________     .sup.1 AS-SS-70  Aeromonas salmonicide - 1970 South Santiam isolate           virulent                                                                      .sup.2 LS-1-74  Vibrio anguillarum - 1974 Lint Slough isolate  fast growe     .sup.3 MSC-2-75  Vibrio anguillarum - 1975 Marine Science Center, Yaquina     Bay isolate  slow grower                                                      .sup.4 AS-Sil-67  Aeromonas salmonicide - 1967 Siletz isolate  avirulent.     Since ASSS-70 autoagglutinates, the nonautoagglutinating, antigenically       similar, avirulent ASSil-67 was used as the indicator antigen instead.   

The remaining fish were subjected to an artificial water-bornefurunculosis challenge. The combined lots not receiving furunculosisbacterin suffered an 85% mortality while the combined lots receiving thebacterin suffered a 72% mortality. The difference was significant at the95% confidence interval.

EXAMPLE 4

Bentonite was added to vaccine preparations in an attempt to increaseantibody titer in the sprayed fish. A late stationary phase culture ofV. anguillarum LS-1-74 was grown at 30° C. in BHI and divided into threealiquat parts. Groups of approximately 30 coho salmon (20/lb.) weresprayed with the following preparations.

1. Uninoculated BHI (control).

2. Late stationary phase culture of V. anguillarum LS-1-74.

3. Late stationary phase culture of V. anguillarum LS-1-74 plus 0.15%bentonite (w/v).

4. Late stationary phase culture of V. anguillarum LS-1-74 plus 0.15%bentonite (w/v) adjusted to a 9.0 pH with 5 N NaOH.

At the end of a 30 day period the geometric mean of the titers were asfollows:

    ______________________________________                                        GROUP            TITER                                                        ______________________________________                                        1                0                                                            2                1:32                                                         3                 1:388                                                       4                1:97                                                         ______________________________________                                    

These titers were at least as good as the titers obtained by theinjection of fish held at 12° C.

EXAMPLE 5

This experiment was conducted to investigate the parameters of the spraypressure necessary to provide satisfactory immunization with Vibriovaccine.

Juvenile coho salmon in groups of 60 were sprayed with the bacterin acidcontrol at a series of pressures with a paint spray and a sandblast gun.The pressures employed were 20, 30, 40, 50, 70 and 90 psi. As a tracer,a red or yellow fluorescent dye was added to the bacterin dispersion. Apressure of 70 to 90 psi was found necessary to provide dye retention onall of the fish sprayed with the sandblaster. At these pressures thepaint sprayer provided from 50 to 88% retention.

Triplicate samples of 20 fish from each group were exposed to Vibrioanguillarum challenge and 10 titers were run from each group. Table 3below gives the geometric mean for titers and the survival rates found 5weeks following treatment. High blood titers and survival rates wereevident at all spray pressures after 5 weeks. Blood antibody titerstaken on an 8 fish sample 4 weeks after the spray immunization treatmentusing a sandblaster with 90 psi showed a geometric mean of 279.

                                      TABLE 3                                     __________________________________________________________________________    Color retention, geometric mean, blood titers and survival rates              for juvenile coho salmon sprayed with Vibrio anguillarum vaccine              and fluorescent pigment at 20, 30, 50, 70 and 90 psi                          with a sandblaster and paint sprayer.                                                         Geometric mean                                                                         Accumulative mortality                                          Percent                                                                            (n = 10) antibody                                                                      3 replicates, 20 fish                                                                     Mean                                     Treatment  color                                                                              titer (5 weeks                                                                         each total 60 fish                                                                        percent                                  group      retention                                                                          after treatment)                                                                       per group   survival                                 __________________________________________________________________________    Control spray                                                                 (No vaccine)                                                                             100  0        59          1.6                                      Vibrio anguillarum                                                            vaccine sprayed with                                                          sandblaster at:                                                               0-1 psi    --   32       0           100                                      3-5 second immersion                                                                     --   7        0           100                                      20 psi     17   588      2           96.6                                     30 psi     66   1,176    2           96.6                                     50 psi     53   388      0           100                                      70 psi     100  1,176    0           100                                      90 psi     100  676      1           98.3                                     with paint sprayer                                                            at:                                                                           20 psi     39   1,024    1           98.3                                     30 psi     60   891      1           98.3                                     50 psi     48   1,024    0           100                                      70 psi     50   588      2           96.6                                     90 psi     88   891      1           98.3                                     __________________________________________________________________________

In addition to the specific embodiments shown various alternatives andmodifications are possible. The vaccine preparations may be modified byusing different killing techniques or incubation periods. Instead ofbentonite other absorbents or cation exchangers may be employed. Variousadjuvants as known to the art may be included or substituted in thevaccine. The spray pressure may be varied up to a maximum which isharmful to the fish and the pattern of spray may be adjusted as appearsnecessary.

The fish may be vaccinated directly in dip nets on a batch basis.Alternatively they may be treated in a continuous manner by transferringthe fish from one holding area to another via a trough, and having aspraying device arranged to spray the fish as they come through thetrough.

We claim:
 1. A method for vaccinating fish against diseases includingVibrio anguillarum, Aeromonas salmonicida, and furunculosis, comprisingdisposing said fish out of water by removal therefrom and into air, andspraying an antigenic composition of killed Vibrio anguillarum, orAeromonas salmonicida, or furunculosis bacterins upon the exterior ofthe fish in the absence of any physical contact between said water andsaid fish.
 2. The fish vaccinating method of claim 1 wherein the saidantigenic composition is dispersed by said spraying within gaseous spaceseparating said spray means from said fish whereby a substantial portionof said fish exterior receives said antigenic composition thereon. 3.The fish vaccinating method of claim 2 wherein said fish is directed tomove through said dispersed antigenic composition whereby vaccinatingsaid fish is in a continuous manner.
 4. The fish vaccinating method ofclaim 1 wherein the antigenic composition contains a small amount of anadjuvant comprising an absorbent of the class exemplified by bentoniteand an anion exchanger.
 5. The fish vaccinating method of claim 1wherein the antigenic composition contains an abrasive powder.
 6. Thefish vaccinating method of claim 1 wherein the spraying employs apressure ranging up to about 90 psi.
 7. The fish vaccinating method ofclaim 6 wherein the pressure employed is from about 70 psi to about 90psi.